Method of determining polymerase activity

ABSTRACT

Method of determining polymerase activity by incubating the polymerase with a template nucleic acid, one detectable mononucleoside triphosphate and one immobilizable nucleoside triphosphate, binding the immobilizable nucleotides to a solid phase and detecting the bound detectable nucleotides and tests based thereon.

The invention adresses a method of determining polymerase activity, areagent and a reagent kit for implementing said method and the use ofsaid method for the detection of HIV. The invention also addresses amethod of determining the inhibitory effect of substances on polymeraseactivities, a method of detecting antibodies to polymerase and a methodof detecting promoter sequences.

Polymerases are enzymes of fundamental importance to living beings. Theyare responsible, for example, for the synthesis of nucleic acids andtheir transformation into other nucleic acids necessary for thesynthesis of proteins. Polymerases are, therefore, found in all types ofcells including, for example, bacteria. Even many viruses code for theirown polymerases. One special representative is a polymerase known asreverse transcriptase (RT).

With the discovery of human pathogenic retroviruses in the last decade(Science (1983), 220:868-871, Proc. Natl. Acad. Sci. USA (1980),77:7415-7419 and Biochem. Biophys. Res. Comm. (1984), 121:126-133), thedetection of the reverse transcriptase (RT), being the typical keyenzyme of this virus family, has gained more and more importance.Numerous improvements of the method of determining RT-activity havesince been described (Virology (1985) 147:326-335). RT-tests arecommonly used in the following 3 situations:

1. characterizing a newly isolated virus as a retrovirus and todifferentiate between different retroviruses,

2. determining the success of a virus isolation from test material of asubject known to be infected with a retrovirus or a subject that may beinfected and

3. clarifying the in-vitro effectivity of chemotherapeutic agentsagainst these enzymatic functions, that are essential for retroviruses.

With respect to the first two of the above mentioned fields ofapplication, the known RT-test is limited in that it involves tedioussample preparation (ultracentrifugation or PEG precipitation,sensitivity may be too low otherwise); as regards the second field ofapplication, the competition by sensitive, less expensive and simplermethods of antigen detection (ELISA); what limits all three of the abovefields of application is the fact that conventional RT-tests use isotopenucleotides thus making special demands on the laboratory (equipment,authorization, personnel, waste disposal).

All methods of detecting enzyme activity of a reverse transcriptase,particularly reverse transcriptase of HIV, that are known fromliterature are based on assays, where a primer, which is complementaryto a partial sequence of a template, is extended by incorporatinglabelled nucleotides. Non-incorporated nucleotides are usually separatedfrom the extended primer that is bound to the template via hydrogenbonds by precipitating the polymer with the aid of trichloroacetic acidor ethanol followed by a filtration procedure or by adsorption of thepolymer to a membrane filter having a positively charged surface (e.g.to DEAE, as described in Journal of Virological Methods 19 (1988),161-168); or by employing any other method suitable to separate anegatively charged polymer from a negatively charged monomer. Thesemeasures are very tedious and require additional handling steps.

WO 90/06373 describes a method which uses immobilized nucleic acids astemplate nucleic acids. A drawback of this method is that sufficientspecific immobilization of this template nucleic acid withoutobstructing the access for enzymes is not possible. The method can,hence, be limited by the available amount of reactive template nucleicacids. Further, the method produces a large number of different nucleicacids for which the binding to the solid phase via the template-DNAgreatly varies as a consequence of different hybridisation conditions.Moreover, reactions at a solid phase generally exhibit poorer kineticsthan do reactions in solutions.

Moreover, it has been found that for the quantitative determination ofHIV-particles, immunoassays for the detection of HIV-antigens are notreliable (New England Journal of Medicine(1989). Vol. 321, 24,1673-1675).

J. Clin. Mircobiology 27 (1989), 7, 1453-1455 and AIDS Research andHuman Retroviruses 5 (1989), 535-540 disclose a method of detectingantibodies to HIV-RT which is based on the determination of polymeraseactivity in the presence of antibodies to HIV. The radioactivelylabelled nucleic acids synthesized by the polymerase are immobilized atmembranes in a non-specific manner. This method is also very tedious.

It was, hence, an object of the invention to provide a quick, simple,more reliable and more sensitive test for polymerase activity. Thisobject has been accomplished by the invention.

The subject matter of the invention is a method of determiningpolymerase activity comprising the following steps:

incubating a polymerase-containing sample with a template nucleic acidand a detectable mononucleoside triphosphate,

separating the synthesized nucleic acid from the detectablemononucleoside triphosphate and

detecting the detectable nucleotides contained in the nucleic acidswhereby

the incubating mixture contains, in addition, an immobilizablemononucleoside triphosphate and

the incubating mixture is brought into contact with the solid phase,said solid phase binding selectively and essentially completely theimmobilizable mononucleoside triphosphate and the nucleic acids in whichthe immobilizable nucleotide is incorporated.

Further subject matters of the invention include reagents forimplementing the method, a method of determining the inhibitory effectof substances on polymerase activity, a method of detecting antibodiesto polymerase and a method of detecting promoter sequences.

Polymerase activity is understood to be the enzymatic activity ofpolymerases to polymerize mononucleoside triphosphates into a nucleotidesequence with the aid of a template nucleic acid. In this process, themononucleotides are added to a nucleic acid sequence and incorporatedinto a nucleic acid strand complementary to a nucleic acid sequence.Depending on the polymerase, the resulting product is either asingle-stranded or a double-stranded nucleic acid.

There are several types of polymerases which can be determined with themethod of the invention. These polymerases are distinguished accordingto substrate and/or polymerization product. Examples includeDNA-dependent RNA-polymerases (type I), such as T3-, T7- orE.coli-RNA-polymerase, DNA-dependent DNA-polymerases (type II), such asKlenow-fragment, RNA-dependent DNA-polymerases (type III), such asreverse transcriptase, and RNA-dependent RNA-polymerases (type IV), suchas Qβ- and Picornavirus-polymerase. In the method of the invention, forexample, viral reverse transcriptases, especially HIV-reversetranscriptase are used as analytes.

In the present invention the term template nucleic acids refers tonucleic acids which function as a substrate of the polymerase or thetype of polymerase to be determined. Substrates for type I and type IIare therefore deoxyribonucleic acids, substrates for type III and typeIV are ribonucleic acids. A deoxyribonucleic acid may also serve as asubstrate for type IIl. To make a distinction from type II, however, thepreferred substrate is a ribonucleic acid.

As regards type II and III polymerases, it is possible to useheteropolymeric as well as homopolymeric nucleic acids as templatenucleic acids. The measurement of in-vitro activity of type II or typeIII-polymerases usually requires the addition of primers.

Testing for type I-polymerases requires the use of a nucleic acid havinga corresponding specific promoter such as the lac-promoter from E. colifor E.coli RNA-polymerase or promoters from T7-phages forT7-RNA-polymerase (Chainberlin and Ryan (1982). The Enzyme, Vol. XV, pp87-108, ). Conversely, the detection of a specific promoter requires aspecific polymerase.

The template nucleic acids for the determination of type IV-polymerasesalso have specific primer structures including terminal-bound proteins,certain secondary structures, etc. These structures depend on thepolymerase and are known to the expert.

Mononucleoside triphosphates are the triphosphates of nucleosidescontaining either natural bases such as adenine, guanine, cytosine anduracil or thymine or artificial bases such as aza- and deaza-analogs ofthe natural bases, for example, 7-deaza-2'deoxyguanosine triphosphate.The term mononucleoside triphosphates refers to monoribonucleosidephosphates or monodeoxyribonucleoside triphosphates depending on thetype of polymerase activity to be determined. Types I and IV requireribonucleotides, whereas types II and III require deoxyribonnucleotides.As regards the reverse transcriptase, it is possible to use both ribo-aswell as deoxyribonucleotides. Deoxyribonucleotides are, however,preferred since the combination of deoxyribonucleotides with ribonucleicacids as template nucleic acids is specific to type III-polymerases.

Detectable mononucleoside triphosphates as defined by the invention aremodified triphosphates. This modification allows a selective detectionof the mononucleotides in the presence of natural mononucleotides, forexample, in photometric, fluorometric, radiometric, enzymatic orimmunological reactions. In a preferred manner, the mononucleosidetriphosphate has a covalently bound non-radioactive chemical group. Thismay be a dye, a fluorescent label or a component of an immunologicreaction such as an antigen, antibody or hapten. Haptens are preferred,with digoxigenin being particularly preferred, because of its highsensitivity. In this context, we refer to the disclosure of EP-A-0 324474 and EP-A-0 254 090.

In addition to the detectable mononucleoside triphosphate, the presentinvention also uses an immobilizable mononucleoside triphosphate.Immobilization of these mononucleotides can be achieved, for example, bycovalently bound chemical groups which have a specific affinity for asolid phase. In a preferred manner, the chemical group is one of thebinding partners of a specific combination. Such combinations are, forexample, antigen-antibody, hapten-antibody, antibody-antibody,vitamin-binding protein, cofactor-enzyme and sugar-binding protein(lectin). A particularly preferred combination is vitamin-bindingprotein, especially biotin-avidin or biotin-streptavidin, biotinrepresenting the chemical group of the mononucleotide bound via alinker. In order to obtain the results intended by the invention, theimmobilizable mononucleoside triphosphate should be different anddistinguishable from the detectable mononucleotide. The method is thenconsiderably less susceptible to interference and less dependent fromthe amount of incorporated nucleotide as compared to the case where themononucleotides are identical. As has been shown for biotin- anddigoxigenin-nucleotides, the respective polymerase must accept thedetectable and immobilizable mononucleoside triphosphate as a substrate.

Any liquid to be tested for its contents of polymerases is a suitablesample for the method of the invention. The method is suitable todetermine polymerases in liquids in preferred concentrations of 10⁻¹² to10⁻⁸ mol/l, particularly preferred 8×10⁻¹¹ to 8×10⁻¹⁰ mol/l (determinedfor RT). Solutions with such concentrations are, for example, obtainedwhen isolating polymerases but are also found in lysates of viruses andbacteria or in supernatants of cell cultures. The lysates can be bothlysates from tissue and from body liquids such as blood. Furtherisolation of the polymerase from lysates is not ncessary. Thepreparation of such samples is, for example, described in Virology(1985) 147:326; PNAS (1980) 77, 7415. Due to its high sensitivity, it isan advantage of the method that in most cases tedious concentrationsteps are no longer required.

In a first step of the invention, a template nucleic acid and at leastone detectable mononucleoside triphosphate and one immobilizablemononucleoside triphosphate are added to the sample. If a polymerase isto be detected whose activity depends upon the presence of a primer, theprimer used is an oligonucleotide or a polynucleotide, which is shorterthan the template nucleic acid and complementary to a part of thetemplate nucleic acid and which, by means of the polymerase, can beextended by a strand segment that is complementary to the templatenucleic acid. The prevailing conditions must allow the polymerase tofully develop its enzymatic activities. These conditions vary dependingon the polymerase but are known to the expert. They include, forexample, the addition of a pH buffer to stabilize the pH value near theactivity maximum of the polymerase or, for example, the presence ofcations necessary for the respective polymerase activity. It is alsopossible to add detergents such as Triton X 100 or complex former suchas EDTA. The optimum temperature is also known to the expert. Thedetection of thermophilic polymerases, e.g. Taq-DNA-polymerase of EP-A-0258 017, can, for example, be carried out at elevated temperatures. Ifthe template nucleic acid is a homopolymer, the mononucleosidetriphosphate must comprise a nucleotide which is complementary to thenucleotide of the template nucleic acid. If polyadenylate is used as atemplate nucleic acid, the triphosphate preferably contains a thymine oruracil residue since the latter can be used to form a nucleic acidstrand that is complementary to polyadenylate.

Moreover, the incubating solution can also contain the non-detectableand/or non-immobilizable mononucleoside triphosphate which is the basisfor the detectable and/or the immobilizable mononucleoside triphosphate.

If the template nucleic acid is a copolymer consisting of all 4mononucleotides, the incubating solution must contain each of thecorresponding 4 mononucleoside triphosphates, regardless of its form, beit detectable, immobilizable or non-detectable or non-immobilizable. Ina preferred case, the concentration of the four mononucleosidetriphosphates is approximately the same and a part of a triphosphate ispresent in its immobilizable form and another part of the sametriphosphate or of another triphosphate is present in its detectableform. The preferred total concentration of each triphosphate is at0.0001 to 10 mmol/l, a particularly preferred concentration rangesbetween 0.001 to 10 mmol/l.

The incubation period for the detection of the reverse transcriptase ina concentration of appr. 10⁻¹¹ mol/l can be terminated after appr. 90min at 35° C. Smaller quantities up to 10⁻¹² mol/l can be detected whenthe incubating period is extended to 24 hours.

Incubation may be carried out in any available pre-incubating vessel,for example a microtiter plate or a tube or vial but also when alreadyin contact with a solid phase to which the immobilizable mononucleosidetriphosphate or the nucleic acids containing the latter are able tobind. In a preferred case, the pre-incubating vessel already containssaid solid phase, and can, hence, bind said compositions. Transfer intoanother vessel for incubation with the solid phase is then no longerrequired.

During a separation step, the nucleic acids, which may have beenproduced due to the polymerase activities and contain at least oneincorporated immobilizable and at least one incorporated detectablemononucleotide, are bound to a solid phase and the liquid phase isremoved.

The solid phase to which the immobilized mononucleoside triphosphate andthe nucleic acids, in which said mononucleoside triphosphate wasincorporated, are bound contains the other binding partner. In aparticularly preferred manner, the solid phase is coated withstreptavidin. The solid phase may be present in the form of a containeror as beads. Typical and preferred vessels are cuvettes and microtiterplates. When beads are used, immobilization requires a separate vesselto contain the incubation mixture unless the preincubation vessel isused.

The solid phase must be able to preferably bind 90% or more of the totalquantity of immobilizable mononucleoside triphosphate and nucleic acidwith the immobilizable nucleotide incorporated therein contained in theincubating solution. For this purpose, the solid phase should contain atleast 90%, preferably more binding sites than the originally availablequantity of immobilizable mononucleotide. Since this quantity is known,the expert can easily determine the necessary surface or, conversely, bymeasuring the binding ability of the solid phase with respect tosolutions containing different quantities of mononucleotides, it ispossible to determine the largest possible quantity of mononucleotidethat is bound by the solid phase. It does, however, not interfere withthe determination if the amount of binding sites at the solid phaseexceeds the quantity of mononucleotide. Streptavidin-coated solid phasesas described in EP-A-0 344578 are particularly preferred solid phases.The use of a specific binding pair ensures that only a minor amount ofnon specifically bound, labelled mononucleoside triphosphate is present.Due to the affinity of the above described binding partners, especiallybiotin/streptavidin, the immobilizable mononucleotides are almostcompletely bound.

In the case of biotin/streptavidin, incubation of the solid phase withthe incubation mixture at 35° C. lasts less than 2 hours. The liquid isthen separated from the solid phase, for example by means of decanting,pipetting or drawing off. Non-incorporated detectable mononucleosidetriphosphate is thus separated from the immobilized detectablemononucleotide. To ensure complete separation, a post-treatment with awash solution may follow. Due the firm binding of the nucleic acidscontaining incorporated immobilizable nucleotides to the solid phase,there is no significant washing-out of the immobilized nucleic acids.Possible wash solutions include, for example, simple salt solution suchas buffered and unbuffered sodium chloride solutions. It is, hence, notnecessary to use wash solutions that maintain hybridization of thetemplate nucleic acids to the newly formed nucleic acids, as is requiredin prior art.

Subsequently, the nucleic acids bound to the solid phase are determinedwith the aid of the also incorporated detectable nucleotides. Thedetermination depends upon the type of detectable nucleotide used. In apreferred case, where the detectable group of the mononucleotide is onecomponent of an immunological reaction, the solid phase is reacted withthe other component of said immunological reaction. This secondcomponent is preferably labelled, e.g. with an enzyme or a fluorescentlabel. Particulary preferred is labelling with enzymes such ashorse-radish peroxidase (POD), alkaline phosphatase or β-galactosidase.In the preferred case the first component is a hapten and the secondcomponent is a labelled antibody to this hapten. In this connection werefer to WO 89/06698 and EP-A-0 209 875. If the label is a direct labelsuch as a dye or a fluorescent dye, the detectable mononucleotide can bedetermined directly either by means of visual or automated analysis, inthe latter case with a photometer or a fluorometer. If an enzyme labelis used, the solid phase is reacted with a substrate for this enzyme andthe reaction is photometrically or fluorometrically monitored.

In the method of the invention, the quantity or the presence of apolymerase or a type of polymerase can be understood from the quantityor the presence of detectable mononucleotide at the solid phase. Aquantitative analysis can be based on a calibration curve obtained withsamples whose contents of polymerases is known.

If the conditions under which the individual polymerases and/or types ofpolymerases incorporate detectable and immobilizable mononucleotidevary, it is possible to discriminate between individual polymerases andbetween types of polymerases. The method of the invention is, hence, aselective procedure allowing, for example, the determination ofpolymerases of the one type in the presence of polymerases of the othertype.

It is an advantage of the method in accordance with the invention thatthe labelled nucleic acid can be separated from the labelledmononucleoside triphosphate in a very simple and complete manner.Moreover, this procedure can be carried out in one single vessel.Transfers into different vessels are no longer required. The use of anon-radioactive assay method has further advantages: the reactionrequires less instrumentation and it is easier to automate and allowsparallel determinations of numerous samples, for example in microtiterplates. The method is highly specific since only immobilizablenucleotides and nucleic acids containing the latter are bound to thesolid phase. Samples with a less-than-maximum degree of puritiy are,hence, also acceptable for the method of the invention. Evendetermination in the presence of bacteria is, for example, possible. Itis not necessary to separate the newly formed strand from the templatenucleic acid when a hapten such as digoxigenin is used as the detectablemononucleotide. The method of the invention is considerably morereliable since the length of the product of the polymerase reaction hasless impact on the result than in the case where only one modifiedmononucleotide is used. When differently modified mononucleotides areused, the amount of immobilizable mononucleotide can be kept low infavor of a quantitative specific binding to the solid phase whereas theamount of detectable mononucleotide can be kept comparatively high for ahigher sensitivity.

In a preferred embodiment of the method of determining reversetranscriptase, the sample is reacted with polyadenylate as a templatenucleic acid, biotinylated dUTP (EP-A-0 063 879) as immobilizable anddigoxigenin-labelled dUTP (WO 89/06698) as detectable mononucleosidetriphosphate and oligodT as a primer. After incubating at appr. 35° C.for 90 min., the mixture is transferred into a streptavidin-coatedcuvette. After incubating for appr. 60 min, the liquid is discarded andthe cuvette is rinsed with a wash solution. Then, a solution ofPOD-labelled antibodies to digoxigenin is added. After a period of appr.60 min, the liquid is again discarded and the cuvette is rinsed oncemore. Then, an ABTS® solution is added and the coloration is measured at405 nm.

Another subject matter of the invention is a reagent for use in themethod of the invention and a template nucleic acid containing at leastone detectable and at least one immobilizable mononucleosidetriphosphate. The reagent is preferably free of polymerase. Thedetectable and the immobilizable mononucleoside triphosphate aredifferent compounds based either on the same mononucleotide, e.g.uridine, or on different mononucleotides, e.g. adenosine (immobilizable)and uridine (detectable). If the template nucleic acid is ahomopolymeric nucleic acid, e.g. polyadenylate, only one type ofmononucleotides is required for the detectable and immobilizablemononucleoside triphosphate, i.e. the one containing the base (in thisexample uridine or thymidine) which is complementary to the nucleotideof the nucleic acid. If the template nucleic acid contains all thenatural bases, the respective complementary mononucleotides must bepresent as triphosphates. In a preferred manner, the reagent alsocontains additives including water as a solvent, pH buffer substances,detergents, stabilizers or co-factors for the polymerase. A pH buffersubstance is included to ensure a pH, alter adding the reagent to thesample, at which the polymerase to be detected, if present, is expectedto be active.

The reagent in the form of an aqueous solution contains the aboveingredients at the following concentrations:

    ______________________________________                                        template nucleic acid                                                                          0.1        to 2000 μg/ml                                  mononucleoside triphosphate                                                                    0.0001     to 10   mmol/l                                    (total) preferably                                                                             0.001      to 10   mmol/l                                    detectable mononucleotide                                                                      0.0001     to 10   mmol/l                                    preferably       0.001      to 1    mmol/l                                    immobilizable mononucleotide                                                                   0.0001 μmol/l                                                                         to 0.1  mmol/l                                    ______________________________________                                    

If a primer is required, it should be present at a concentration of 10ng/ml to 10 mg/ml, or preferably at a concentration of 10-2000 μg/ml. Ifbiotin-dUTP is used as an immobilizable mononucleotide anddigoxigenin-dUTP as a detectable mononucleotide, said two substances areused in the reaction at a molar ratio of 1:1 to 1:50000: for a reversetranscriptase the preferred molar ratio is 1:5 to 1:50, particularlypreferred 1:10 to 1:30 (bio:dig). If nucleotides are used which aremodified in a different way, an expert can optimize the valuesaccordingly. The values can also be optimized in dependency upon thepolymerase activity and the number of binding sites at the wall.Further, the reagent can also be used in the form of a concentratedsolution which has to be diluted prior to use. Storage as a lyophilisateis also possible. In the latter case, the reagent is reconstituted withthe aid of a solution.

Yet another subject manner of the invention is a reagent kit for thedetection of polymerase activity. The kit contains the following inseparate containers:

template nucleic acid, at least one detectable and at least oneimmobilizable mononucleoside triphosphate

a vessel to selectively and essentially completely bind theimmobilizable mononucleotide, and

wash solutions

If necessary, a primer is included in the reagent kit (preferably in amixture with the template nucleic acid). Aside from this reagent, thekit further contains a vessel necessary for carrying out the method anda wash solution. Moreover, a preferred embodiment of the kit containsreagents for the detection of the detectable mononucleotide. If, forexample, an immunological combination for binding a label to thedetectable mononucleotide is used, this kit provides the correspondinglabelled partner of the component of the immunological combination whichis bound to the detectable mononucleotide. When an enzymatic label isused, the kit also comes with an enzyme substrate and, if necessary, therequired pH buffer substances. It is an advantage of the reagent kit ofthe invention that all reagents are stable and require relatively littlestorage space and their use and application is much easier as comparedto reagents known in prior art.

A preferred embodiment of a reagent kit contains:

a) a container with template nucleic acid, digoxigenin-labelledmononucleoside triphosphate, biotin-labelled mononucleosidetriphosphate, pH-buffer, detergent, complex former, co-factors, saltsand anti-oxidizing agents

b) streptavidin-coated cuvette, tube, pellets or microtiter plate

c) wash solution

d) solution of a POD-labelled antibody to digoxigenin

e) A POD substrate, for example,2,2'-azino-bis-13-ethylbenzothiazoline-6-sulfate] (ABTS®) in buffersolution

For the detection of type II or type III polymerases a), in a preferredmanner, also contains a primer. All components of the reagent kit arestable, have extended storage stability, and both their manufacture anddisposal are inexpensive and simple.

The method of the invention for detecting a polymerase activity can beemployed in any field where such a polymerase activity revealsinformation on the composition of a sample. This is the case, forexample, in the detection of polymerases in samples such as body fluidsor processed enzyme isolates, further in the detection ofpolymerase-containing micro-organisms, particularly bacteria and virusesto characerize unknown polymerases or micro-organisms and to testsubstances for their possible inhibitory effect on polymerases. Thedecrease of the known polymerase activity upon addition of the substancethen serves as a measure of inhibition and indicates a possibletherapeutic use of the substance as an anti-bacterial or anti-viralagent.

Another subject manner of the invention is a method of detecting HIV,said method being based upon the method of determining polymerases. Thena lysate of a sample in which HIV is expected to be present is prepared.

a template ribonucleic acid, at least one detectable and oneimmobilizable mono-deoxyribonucleoside triphosphate and additives areadded to this lysate,

after incubation, the mixture is transferred into a vessel whichselectively and essentially completely binds the immobilizablemononucleotide and immobilizable nucleotide incorporated.

excess detectable mononucleoside triphosphate is separated and

the bound detectable mononucleotide is detected.

The first incubation is preferably carried out in the presence of aprimer since the HIV-RT is a primer-dependent type-Ill polymerase. Theincubation of the mixture could also take place in the vessel whichitself is capable of binding.

This method applies analogously to the detection of other virusesconnected to a polymerase. In this case a virus isolate, e.g. a pellet,may serve as sample.

Yet another subject matter is a method of detecting an antibody to apolymerase by

incubating the sample containing the antibody to be detected with atemplate nucleic acid, a detectable mononucleoside triphosphate and thepolymerase,

separating the synthesized nucleic acid from the detectablemononucleoside triphosphate and

detecting the detectable nucleotides contained in the nucleic acid,whereby

the incubation mixture contains an additional immobilizablemononucleoside triphosphate and

the incubation mixture is or is brought in contact with a solid phasewhich is able to selectively and essentially completely bind theimmobilizable mononucleoside triphosphate and the nucleic acid in whichthe immobilizable mononucleotide is incorporated.

This process is based on the detection of polymerase activity accordingto the above invention. The reaction mixture thereof exhibits a certainpolymerase activity. Said polymerase activity is compared the onepresent after addition of the sample which is expected to contain anantibody to this polymerase. If an antibody that is directed againstthis polymerase is present, the activity will be lower as compared tothe activity with no antibody present. The sample material used may beblood, serum, an isolate, a cell culture supernatant or the like.

Therefore, the quantity and the type of reagents used essentiallycorresponds to those used in method of detecting polymerases. Theconcentration of the polymerase also depends upon the quantity of theantibody to be detected. However, the expert can find a suitabledilution by running a few tests for this purpose beforehand. Thedetection range for the method of detecting a polymerase according tothe invention may serve as a reference in this case.

Further, it can be advantageous to eliminate the polymerase activityoriginally present in antibody-containing sample prior to adding thepolymerase. Other common reaction conditions are known to the expert,e.g., from J. Clin. Microbiology. (1989). Vol. 27, 7, pp. 1453-1455 andfrom AIDS Research and Human Retroviruses. (1989). Vol 5, pp. 535-540.

The detection of antibodies to type II or type III polymerases requiresan additional corresponding primer.

In a preferred embodiment of the method of detecting antibodies toHIV-RT, the template nucleic acid, biotinylated dUTP (EP-A-0 063 879) asimmobilizable mononucleoside triphosphate and digoxigenin-labelled dUTP(WO 89/06698) as detectable mononucleoside triphosphate and a definedquantity of polymerase, e.g. reverse transcriptase, which is obtainedfrom virus lysate or, preferably, by means of genetic engineering, aredirectly incubated with the serum to be analyzed for appr. 60-90 min at37° C. After incubation the mixture is transferred into astreptavidin-coated cuvette. After incubation for appr. 60 min, theliquid is discarded and the cuvette is rinsed with a wash solution. Thena solution of POD-labelled antibodies to digoxigenin is added. Theliquid is again discarded after appr. 60 min and the cuvette is againrinsed. Then, an ABTS® solution is added and the coloration is measuredat appr. 405 nm.

In the method of the invention, the quantity or the presence ofdetectable mononucleotide at the solid phase follows from the quantityor the absence of the antibody to the polymerase. A quantitativeevaluation is, for example, possible with the aid of a calibration curveobtained with samples that contain a known amount of antibody.

If the conditions under which the individual polymerases or types ofpolymerases incorporate detectable and immobilizable mononucleotidevary, it is possible to differentiate between antibodies to individualpolymerases and between antibodies to types of polymerases. The methodof the invention is, hence, a selective method. It is, for example,possible to determine antibodies to polymerases of the one type in thepresence of antibodies to the other type.

The method of the invention can, for example, be used for tile detectionof an infection with a micro-organism which contains a polymerase orcontains information for the production of a polymerase, e.g. virusessuch as the HIV. The method has the same advantages as the method ofdetecting polymerase activity, i.e. it is easy, rapid, reliable and moresensitive than the known methods. It is an additional advantage of themethod that is does not require tedious IgG-preparations. Serum can bedirectly used as a sample.

Yet another subject matter of the invention is a method of detecting apromoter sequence comprising the following steps:

incubation of a sample containing the promoter sequence to be detectedwith a detectable mononucleoside triphosphate and a polymerase whichrequires the promoter sequence to initiate the polymerase reaction,

separation of the synthesized nucleic acid from the detectablemononucleoside triphosphate and

detection of the detectable nucleotides contained in the nucleic acid,whereby

the incubation mixture contains an additional immobilizablemononucleoside triphosphate and

the incubation mixture is or is brought in contact with a solid phasewhich is able to selectively and essentially completely bind theimmobilizable mononucleoside triphosphate and the nucleic acid in whichthe immobilizable mononucleotide is incorporated.

This method is also based on the determination of polymerase activity inaccordance with the invention. However, this method is distinguishedfrom the latter in that a given amount of polymerase is added whereastemplate nucleic acid is not added in addition to the nucleic acid whichis present in the sample to be detected and may contain the promotersequence. If a promoter is present that is able to initiate thepolymerase which was added, the polymerase reaction will occur andlabelled nucleic acids can be detected. Promoters which can be detectedinclude the SP6. the T7- and the T3-promoters. The respectivepolymerases, i.e. the SP6-RNA-polymerase, the T7-RNA-polymerase and theT3-DNA-polymerase, are then used. The test is specific for therespective promoter. In molecular biology, such tests are advantageouslyused, for example, in the detection of nucleic acid constructs. Thepreferred sample is a DNA isolate.

The concentration values and the conditions for the individual reagentsbasically depend upon the values given in the method of determiningpolymerase in accordance with the invention.

In a preferred embodiment of the invention, a sample is reacted withbiotinylated dUTP (EP-A-0 063 879) as immobilizable anddigoxigenin-labelled dUTP (WO 89/06698) as detectable mononucleosidetriphosphate and with a polymerase which is specific for the promoter.After incubation at appr 35° C. for 90 min, the mixture is transferredinto a streptavidin-coated cuvette. After incubation for appr. 60 min.the liquid is discarded and the cuvette is rinsed with a wash solution.Then a solution of POD-labelled antibodies to digoxigenin is added.After appr. 60 min. the liquid is again discarded and the cuvette isrinsed once more. Then, an ABTS® solution is added and the coloration ismeasured at appr. 405 nm.

In the method of the invention, the strength or the presence of apromoter for the polymerase or the type of polymerase follows from thequantity or the presence of detectable mononucleotide at the solidphase.

If the conditions for incorporating detectable and immobilizablemononucleotide differ for the individual polymerases or polymerasesubtypes, it is possible to differentiate between individual promoters.The method of the invention is, hence, a selective method. It is, forexample, possible to determine promoters of one polymerase subtype inthe presence of promoters of another polymerase subtype. Eukaryotepolymerases of type I exist in different subtypes (RNA-pol I, II, IIl)which recognize different promoter types. And each particular polymerasesubtype in rum recognizes, in dependency upon additional factors,different promoters.

Yet another subject matter of the invention is a reagent kit for thedetection of antibodies to a polymerase. Said reagent kit contains atemplate nucleic acid, at least one detectable and at least oneimmobilizable mononucleoside triphosphate and a polymerase against whichthe antibody to be detected is directed. Preferred further componentsare additives and, optionally, a vessel or any other solid phase whichselectively and essentially completely binds the immobilizablemononucleoside triphosphate and the nucleic acids containing the latter.In a preferred manner, at least the polymerase is available in aseparate container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percentage of RT-inhibition (abscissa) byanti-HIV-positive and anti-HIV-negative sera (1:40) for two differentvirus lysate concentrations.

FIG. 2 shows the percentage of RT-inhibition (abscissa) byanti-HIV-positive and anti-HIV-negative sera ( 1: 40) for recombinant RT(appr. 1:27000).

n in FIGS. 1 and 2 represents the number of sera.

The following examples explain the invention in greater detail:

EXAMPLE 1 Detection of HIV a) Preparation of pelleted HIV-virus

2-3 ml of cell-free supernatant of HIV-virus cultures or virus isolationsolutions are obtained. A subsequent ultracentrifugation at 20 000 g fora period of 2 hours leads to the formation of a pellet of cellularorganelles and (in a positive case) virus particles.

b) Lysis of the pelleted virus

The pelleted virus is disolved in 40 μl lysis buffer. Lysis buffer:

1,4-dithioerythrite (DTT, Merck, Darmstadt) 2.5 mM;

EDTA (Merck, Darmstadt) 0.75 mM;

KCl (Merck, Darmstadt) 80 mM;

Tris HCl (Boehringer Mannhelm) 50 mM; pH 8.0 (22° C.)

Triton®X-100 (Sigma, Saint Louis) 0.5%

Alternatively, it is also possible to add 5 μl of an eightfoldconcentrated lysis buffer to 35 μl virus containing supernatant.

At 2° C. this virus lysate can be stored for about one week withoutsignificant decrease in activity (protect lysate from drying out).

c) Incubating/polymerase reaction

20 μl of a solution of the following composition are added to the abovelysate:

DTT 10 mM;

KCl 290 mM:

MgCl₂ 30 mM;

dTTP 8.3 μM;

Biotin-16-dUTP (Boehringer Mannhelm, Cat. No. 1 093 070) 125 nM;

DIG-11-dUTP (Boehringer Mannhelm, Cat. No. 1 093 088) 2.5 μM;

Polyadenylic acid. Pentadecathymidylic acid (Poly A)·(dT)₁₅,

(Boehringer Mannhelm, Cat. No. 108 677) 700 U/l, [35 μg/ml];

TrisxHCZ 50 mmol/1. pH 8.0 (22° C.)

The mixture is incubated at 35° C. for a period of 90 min. to 24 hoursand even longer depending on the desired test sensitivity.

d) Isolation of the reaction products

After incubating, the reaction mixture is transferred into the wells ofa streptavidin-coated 96-well-ELISA plate (manufactured according toEP-A-0 344 578, capacity appr. 20 ng biotin/ml, i.e. in 60 μl appr. 1.2ng biotin) and then incubated for 60 min. at 35° C.

e) Washing

The wells are then washed 5 times with a wash solution (sodium chloride0.85 mol/l). The solution still remaining in the wells is then removedby tapping on the plate.

f) Incubation with POD-labelled anti-digoxigenin antibodies

200 μl of a conjugate solution of the following composition are pipettedinto each of the wells: anti-digoxigenin-POD (Fab fragments, Cat. No. 1207 733 Boehringer

Mannhelm) 200 U/l;

Tween 20 0.5 %

Sodium chloride 0.85 M in 0.1 mol/l sodium phosphate buffer pH 7.5.

The conjugate solution is decanted after 60 min. and the wells arewashed 5 times with a wash solution (NaCL 0.85 M).

g) Colorimetric reaction

200 μl substrate solution (ABTS®(2'2-Azino-di-[3-ethylbenzthiazolinsulfonate (6))][sic] 1 g/l in ABTS®buffer (Cat. No. 1 112 597. Bothringer Mannheim) are pipetted into eachof the wells. After allowing the reaction to occur for another 60 min.at 35° C., the coloration is measured in a photometer at 405 nm against450 nm.

h) Sensitivity of the tests

The sensitivity of the method of the invention was tested with serialdilutions of aliquots of a positive control (lysate of pelletedHIV-I-particles from appr. 2 ml supernatant of a well producingcontinuous culture of permanent T cells).

                  TABLE 1                                                         ______________________________________                                        Virus dilution                                                                            OD 405 nm Virus dilution                                                                             OD 405 nm                                  ______________________________________                                        1:5         >2.500    1:25         1.900                                                  >2.500                 >2.500                                                 >2.500                 >2.500                                                 >2.500                 >2.500                                                 >2.500                 1.847                                                  >2.500                 1.691                                                  >2.500                 >2.500                                                 >2.500                 1.935                                      1:50        0.730     1:125        0.602                                                  0.868                  0.633                                                  0.793                  0.599                                                  0.880                  0.613                                                  0.812                  0.506                                                  0.838                  0.496                                                  0.769                  0.453                                                  0.990                  0.455                                      1:250       0.350     1:500        0.243                                                  0.348                  0.228                                                  0.340                  0.274                                                  0.404                  0.252                                                  0.307                  0.247                                                  0.308                  0.230                                                  0.242                  0.163                                                  0.245                  0.147                                      ______________________________________                                        Negative control                                                                          OD 405 nm Blank        OD 405 nm                                  ______________________________________                                        1:2         0.130                  0.072                                                  0.160                  0.075                                                  0.138                  0.068                                                  0.119                  0.090                                                  0.133                  0.083                                                  0.187                  0.073                                                  0.121                  0.065                                                  0.183                  0.071                                      ______________________________________                                         OD = Optical density, measured at 405 nm against 450 nm.                      Blank = Optical density of the nonreacted substrate.                     

The above listed measured values are obtained in a minimum of fourexperiments carried out on different days. Between the tests thereaction mixtures used were stored at -20° C. At least two batchesprepared on different days were used. A maximum signal (OD>2.5) occuredregularly up to a dilution of 1:5 in some test runs even up to adilution of 1:25. With a continuous decrease of the signal and asimultaneous increase of dilution, the detection limit of the reversetranscriptase is reached at a dilution of appr. 1:500 after a reactiontime of 90 min. For a reaction time of 24 hours, the limit is at adilution of 1:3000 to 1:7500 (6 to 15 times more sensitive). Thedeviations between the OD-values in the different test runs aretypically at a factor smaller than 2. Compared to a conventional method,the new test with a reaction time of 90 min. is at least as sensitive asconventional methods and is distinguished by a more simple handling.Further, as compared to conventional methods, this specifity of themethod in accordance with the invention allows extending the reactiontime to at least 24 hours and, hence, obtaining a considerably highersensitivity. Even samples that are massively contaminated withhemoglobin from lysated erythrozytes or with bacteria do not show blankswhich exceed the limit. In a direct comparison of various virusisolation solutions (pellet of appr. 3 ml supernatant), the new methodconvinced with its high reliability.

EXAMPLE 2

Method of detecting reverse transcriptase and testing samples for theirpossible inhibitory effect on polymerases

As in example 1, this test is based on a test for reverse transcriptase.

a) Sample preparation

The samples to be tested for the presence of inhibitors are usuallydisolved in concentrations of 1 to 10 mg/l in pure DMSO(dimethyisulfoxide). These stock solutions are the basis for dilutions(usually 20- to 200-fold diluted) by diluting with RT-buffer (50 mMTris-HCl, pH 7.9:50 mM KCl; 5 mM MgCl₂ ; 1 mM DTT).

b) Incubation

The test is carried out directly in the streptavidin-coated96-well-ELISA plates (manufactured according to EP-A-0 344 578).

The following solutions are sucessively pipetted into the wells of theplate:

20 μl diluted sample solution or RT-buffer for positive control

20 μl RT-buffer

20 μl template/primer complex consisting of 1 μg RNA-template(HIV-LTR-5'-gag fragment) and 21.3 ng 18-mer DNA-primer of the followingsequence: SEQ ID NO 1 (5'-GTCCCTGTTCGGGCGCCA-3'): after incubating bothcomponents as a mixture in a 50-fold concentrated form at 66.5° C. in awater bath and cooling the mixture slowly down to 30° C., said mixtureis 10-fold diluted with RT-buffer.

20 μl HIV-1 RT is reached according to a known process cloned andexpressed in E.coli purified to homogenity (Heterodimer, p66/p51,Science (1986), 231:1289-1291) and dissolved in a concentration of 1μg/ml in RT-buffer.

20 μl reaction starter solution consisting of

RT-buffer (see above)

5 μM dATP

5 μM dCTP

5 μM dGTP

5 μM Dig-11-dUTP

0.25 μM Biotin-16-dUTP

0.1% Nonidet® P 40 (Pierce, Cat. No. 28324)

The HIV-LTR-5'-gag RNA-fragment of 1078 bases in length, which serves asa template, was prepared according to known procedures by in-vitrotranscription with the aid of a T7-RNA-polymerase.

The mixture is then at 37° C. incubated for 1 hour in a water bath in amicrotiter plate (capacity for binding biotin appr. 20 ng/ml, i.e. in100 μl appr. 2 ng biotin) with a flat bottom. This microtiter plate wasmanufactured in accordance with EP-A-0 344 578. The microtiter plate iscovered with an adhesive foil.

c) The well is washed 5 times. each time with 0.3 ml of a wash solution(PBS without Ca and Mg (Cat. No. 210 340 Boehringer Mannheim), Tween 20(Cat. No. 170-6531, Bio-Rad) 0.05 % (vol/vol)).

d) 100 μl anti-digoxigenin-POD (Fabfragments, Cat. No. 1 207 733Boehringer Mannheim) contained in a solution of 100 mM Tris-HCl, pH 7.9;150 mM NaCl are added into each well. Incubation is carried out for 30min. at room temperature.

e) Each well is washed 5 times with 0.3 ml wash solution for eachwashing step.

f) 100 μl of the following solution are added into each well: ABTS® 1g/l in ABTS®-buffer (Cat. No. 1 114 497 Boehringer Mannheim)Subsequently, the solution is incubated under shaking for 20 to 30 min.at room temperature and the coloration is then measured at 405 nm in anELISA-photometer (MR 5000, Dynatech, reference filter 630 nm). Apolymerase inihibitor is present if the coloration at 405 nm is sloweddown or missing.

EXAMPLE 3 Detection of specific DNA-dependent RNA-polymerase activityand specific promoter sequences for RNA-polymerases on DNA

Three DNA-fragments, which contain the promoter for either theSP6-RNA-polymerase, the T7-RNA-polymerase or the T3-RNA-polymerase, arein transcription reactions incubated with a mixture of ribonucleotides.The latter also contains Dig UTP and Bio UTP and eitherSP6-RNA-polymerase or T7-RNA-polymerase. In both assays where promoterand polymerase specifically match, the nucleotides are, complementary tothe DNA, incorporated in an RNA-chain. After incubation the reactionsolutions are transferred into the wells of a streptavidin-coated 96well-ELISA plate. Incorporated and non-incorporated biotin residues willbind to the streptavidin. An RNA-polymerase activity can be detected bydetecting the digoxigenin-nucleotides or the digoxigenin-haptens, whichare covalently bound to the biotin-nucleotides with the aid ofanti-digoxigenin antibodies. Correspondingly, it is possible to detect aspecific promoter for a polymerase.

    ______________________________________                                        a) Transcription reaction:                                                    ______________________________________                                        1    μl  template-DNA (100 ng/μl),                                                                         100  ng                                    2    μl  Dig-Bio-Nucleotide-Mix 10 ×                                 0.5  μl  RNAsin (50 U/μl, Cat. No. 799 017,                                                                25   U                                                 Boehringer Mannheim),                                             2    μl  transcription buffer 10 ×                                   x    μl  Phage-RNA-polymerase   (20  U)                                    ad 20                                                                              μl  H.sub.2 O                                                         20   μl                                                                    ______________________________________                                    

Template-DNAs

Plasmides containing the appropriate promoter sequences were cleaved byrestriction enzymes and electrophoretically separated on an agarose gel.DNA-fragments which contained a promoter were eluted from the gel andused as template-DNAs.

a) Template with SP6-promoter

pSPT18 plasmid (Cat. No. 909 793. Boehringer Mannheim) digested with EcoR I+Ssp I, 951 bp-fragment isolated, length of transcript: 58 b

b) Template with T7-prompter

pSPT18 plasmid (Cat. No. 909 793, Boehringer Mannhelm) digested withHind III+Ssp I, 2204 bp-fragment isolated, length of transcript: 61 b

c) Template with T3-promoter (control)

pT3T71ac plasmid (Cat. No. 1 014 692, Boehringer Mannheim) digested withEco RI+Sca I, 1815 bp-fragment isolated, length of transcript: 60 b

    ______________________________________                                        Dig-Bio-Nucleotide-Mix 10 ×:                                            5      μl   100 mM ATP        10   mM                                      5      μl   100 mM GTP        10   mM                                      5      μl   100 mM CTP        10   mM                                      3.25   μl   100 mM UTP        6.5  mM                                      8.75   μl   1 mM Bio-11-UTP   175  nM                                      16.5   μl   10 mM Dig-11-UTP  3.3  mM                                      6.5    μl   H.sub.2 O                                                      50     μl                                                                  Transcription buffer 10 ×:                                              400    μl   1M Tris/HCl pH 7.9                                                                              400  mM                                      60     μl   1M MgCl.sub.2     60   mM                                      100    μl   1M DTT            100  mM                                      20     μl   1M Spermidin      20   mM                                      420    μl   H.sub.2 O                                                      1      ml                                                                     ______________________________________                                    

Phage-RNA-polymerases

SP6-RNA-polymerase, Cat. No. 810 266, Boehringer Mannheim

T7-RNA-polymerase, Cat. No. 881 767, Boehringer Mannheim

As DNA with a spermidin concentration exceeding 4 mM can precipitate,the 10 x-transcription buffer, was added immediately before the additionof the enzymes [Krieg, P. A., Melton, D. A. (1987) Meth. Enzym. 1551. H₂O was treated with DEPC (diethylpyrocarbonate) to inactivate RNAses.

Incubation for 2 hours at 30° C. [after Krieg, P. A. (1990): Nucl. AcidsRes. 18, 6463].

b) Binding of the transcripts to the streptavidin in microtiter plates

The transcription reactions were filled up to 100 μl with 80 μl TE (10mmol/l TrisHCl, 1 mmol EDTA, pH 8.0). Portions of 20 μl of the dilutedtranscription reactions were pipetted into the flat-bottom wells of astreptavidin-coated 96-well-Elisa plate (manufactured according toEP-A-0 344 587) and 180 μl of a 0.05 % (vol/vol) Tween 20 solution wereadded. During all following binding and reaction steps, the plates werecovered with lids (Nunc, Cat No. 263339).

Binding is carried out at 37° C. under shaking for 1 hour.

Non-bound reaction components are removed in 6 washing steps with 300μlof a 0.9% NaCl-solution used in each step.

c) Detection with anti-digoxigenin-antibody conjugate and color reaction

As described with respect to example 1,200 μl anti-digoxigenin-POD (200U/I, Cat. No. 1207733 Boehringer Mannheim) are added. After 60 minutes,6 washing steps are carried out, using 0.9% NaCl solution in each step.As was also desribed with respect to example 1, 200 Ill substratesolution (ABTS®) are incubated in the wells at 37° C. for 5 to 60minutes and the coloration is measured in a photometer at 405 nm.

EXAMPLE 4 Detection of antibodies to HIV-RT in patient sera, saidantibodies having an inhibitory effect on the funtion of the RT

This detection serves as a prognostic factor for an HIV infection andmay serve to distinguish between different variants of HIV (HIV 1 andHIV 2).

10 μl diluted serum (1:10 in lysis buffer, see example 1) are added to30 μl polymerase (obtained from virus lysate or genetically engineeredreverse transcriptase), diluted in 1xconcentrated lysis buffer, andincubated for 60-90 minutes at 35° C. 20μl of the reaction solution (asdescribed under c in example 1) are then added and incubated at 35° C.for 24 hours. The remaining procedure corresponds to the one describedin example 1. If the sample contains antibodies that inhibit thefunction of the reverse transcriptase, the signal will either besuppressed or partly reduced. Serum sample without RT-inhibitingantibodies will produce a signal that corresponds to the amount of RTused.

a) Pre-tests:

Several tests were carried out to determine optimal serum- and virusconcentrations. In these tests, 6 HIV-I-positive and HIV-negative seradiluted 1:32 and 1:64, respectively, were tested with a standard viruslysate diluted 1:250 (lysated HIV-1 from appr. 1 ml supernatant of awell-producing cell culture of HIV-1 infected H9-cells). In anothertest, three additional HIV-1 -positive and HIV-negative sera diluted toconcentrations of 1:32, 1:64 and 1.128, respectively and standard viruslysate dilutions of 1:125, 1:250 and 1:500, respectively, were tested.Additional tests with different dilutions of a preparation ofgenetically engineered (recombinant) RT (manufactured by Mikrogen,Munich, FRG) were also carried out. The optimal polymerase dilution wasfound with the aid of sera whose RT-inhibiting capacities had beencharacterized before the tests were started. Dilutions of appr. 1:25000of recombinant RT haven proven to be the most suitable.

The resulting values showed a significant inhibition of the RT functionwith all HIV-1 positive sera whereas the RT function with HIV -negativesera was not impaired. The relative signal suppression was mostpronounced at serum dilutions of 1:32 and 1:64. The ideal dilution ofour standard virus lysate was found to range between 1:125 and 1:250.

b) Validation of the test principle with patient sera.

Based on the experiences from the pre-tests, 34 HIV-1-positive and 26HIV-negative sera, each diluted 1:40. were tested in two batches, onewith a virus lysate diluted 1:125 and the second with a virus lysatediluted 1:250. The results are listed in FIG. 1. In order to furthervalidate the test principle, a larger number of sera (98anti-HIV-l-positive sera and 76 anti-HIV-negative sera) has been testedfor inhibiting antibodies with recombinant RT (dilution appr. 1:27000).The total result obtained with the recombinant RT is listed in FIG. 2.

c) Test to establish the prognostic value of RT inhibiting antibodies.

From some of the 132 anti-HIV-1-positive sera, we knew the clinicalcondition of the patient (asymptomatic LAS/ARC, AIDS) and from some weknew the test result obtained with the p24-antigen-ELISA (manufacturedby Abbott Laboratories, North Chicago, USA) with serum or plasma. In athird group, the reaction pattern in the Western blot (WB) can be usedto differentiate between diffrerent conditions: shortly afterseroconversion (early infection), patients with insignificant antibodyprofile, beginning drecrease of the anti-p24 level and late stage. Totest the prognostic value of RT-inhibiting serum antibodies, theproportion of the sera were RT-inhibition exceeded 50% and the meanRT-inhibition were calculated in a comparison of all groups (see Table2).

                  TABLE 2                                                         ______________________________________                                        Prognostic value of RT-inhibiting antibodies in comparison                    to acknowledged prognostic parameters (p24-antigenemia,                       clinical rating, WB-antibody profile).                                                        patients corresponds                                                          with     to %       average                                                   RT-      with       RT-                                               number  inhibition                                                                             RT-inhibition                                                                            inhibition                                        tested  >50%     >50%       in %                                      ______________________________________                                        p24-antigen                                                                             15        10       67       60,9                                    positive                                                                      p24-antigen                                                                             38        34       89       77.3                                    negative                                                                      asymptomatic                                                                            29        25       86       79.1                                    LAS/ARC   10        9        90       78.3                                    AIDS      13        7        54       44.4                                    early infection                                                                          5        2        40       44.0                                    WB normal 97        79       81       72.6                                    weak anti-p24                                                                            9        6        67       65.5                                    WB late stage                                                                           16        7        44       45.2                                    ______________________________________                                    

EXAMPLE 5 Detection of DNA-dependent DNA-polymerase activity

Klenow-polymerase is incubated in a random primed labelling reactionwith a thermally denatured DNA fragment and a mixture of hexamersconsisting of hexamers of all possible sequences that may serves asprimers. In addition to unlabelled nucleotides, the polymerase alsoincorporates DIGdUTP and BiodUTP. The double-labelled DNA so produced bypolymerase activity can in turn be quantitatively determined instreptavidin-coated ELISA plates.

a) Reaction mixture

2 μb of a DNA fragment of 356 bp in length are for 10 minutes and at 95°C. denatured in 15 μl H₂ O and then immediately placed on ice. Thefollowing is pipetted to these 15 μl:

2 μl Hexanucleotide Mixture in 10x reaction buffer (from Random PrimedDNA Labelling Kit, Cat. No. 1004760, Boehringer Mannheim)

2 μl 10x nucleotide-Mix

1 μl Klenow-enzyme (from Random Primed DNA Labelling Kit, Cat. No.1004760, Boehringer Mannheim)

    ______________________________________                                        10 × Nucleotide-Mix:                                                                    dATP        2       mM                                                        dCTP        2       mM                                                        dGTP        2       mM                                                        DTTP        1.75    mM                                                        DIGdUTP     0.25    mM                                                        BiodUTP     125     nM                                        ______________________________________                                    

The reaction mixture is incubated for 3 hours at 37° C. and then diluted1:10 with TE buffer (10 mM Tris, I mM EDTA).

A control reaction is subject to an identical procedure except that themixture does not contain BiodUTP.

b) Binding of the reaction products to streptavidin in microtiter platesand detection thereof

Portions of 2 μl and 6 μl of the diluted reaction mixture containing 20ng and 60 ng, respectively, of the DNA fragment used to the labellingreaction, contained in 200 μl TE-buffer, are transferred intostreptavidin-coated microtiter plates and incubated for one hour at 37 °C.

The plates are then washed three times with 300 μl PBS-buffer with 0.5 %(vol/vol) Tween 20.

Subsequently, 200 μl anti-digoxigenin-POD (150 mU/ml, Cat. No. 1207733,Boehringer Mannheim) are added. After one hour three more washing steps,each with 300 μl PBS buffer with 0.5% (vol/vol) Tween 20, are carriedout. Corresponding to the preceding examples, 200 μl ABTS®-substratesolution are then added and the coloration is measured at 405 nm alteran incubation time of 30 min.

    ______________________________________                                        Result                                                                        diluted reaction        diluted reaction                                      mixture                 mixture                                               without BiodUTP         with BiodUTP                                          2 μl   6 μl       2 μl                                                                              6 μl                                        ______________________________________                                        -0.013    0.073         0.285  1.094                                          Sequence protocol:                                                            SEQ ID NO: 1                                                                  Length of sequence:                                                                       18 bases                                                          Type of sequence:                                                                         nucleotide                                                        Type of strand:                                                                           single strand                                                     Topology:   linear                                                                        GTCCCTGTTC GGGCGCCA                                               ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCCCTGT TCGGGCGCCA18                                                     

We claim:
 1. Method for detecting polymerase activity in a samplecomprising the steps of:a) combining a sample suspected of containing apolymerase with a template nucleic acid and mononucleoside triphosphatesto form an incubation mixture, wherein said mononucleoside triphosphatesinclude a detectable mononucleoside triphosphate and a separateimmobilizable mononucleoside triphosphate, wherein said immobilizablemononucleoside triphosphate is now used as a detectable label, b)incubating said incubation mixture to incorporate the detectablemononucleoside triphosphate and the immobilizable mononucleosidetriphosphate into a newly formed nucleic acid, c) contacting saidincubation mixture with a solid phase which selectively binds theimmobilizable mononucleoside triphosphate and the newly formed nucleicacid in which the immobilizable mononucleoside triphosphate isincorporated, d) separating said newly formed nucleic acid from anyunincorporated detectable mononucleoside triphosphate, and e) detectingthe detectable mononucleoside triphosphate incorporated into the newlyformed nucleic acid as a measure of the polymerase activity.
 2. Methodaccording to claim 1, wherein the template nucleic acid is a DNA whichcontains a promoter which is specific for the polymerase and themononucleoside triphosphates are ribonucleoside triphosphates.
 3. Methodaccording to claim 1, wherein the template nucleic acid is a DNA and themononucleoside triphosphates are deoxyribonucleoside triphosphates. 4.The method according to claim 3, further comprising adding anoligonucleotide to the incubation mixture, wherein said oligonucleotideis complementary to a part of the template nucleic acid.
 5. Methodaccording to claim 1, wherein the template nucleic acid is an RNA andthe mononucleoside triphosphates are deoxyribonucleoside triphosphates.6. Method according to claim 1, wherein the template nucleic acid is anRNA which contains a primer structure and the mononucleosidetriphosphates are ribonucleoside triphosphates.
 7. Method according toclaim 1, further comprising adding a polymerase inhibitor to theincubation mixture formed in step a).
 8. Method according to claim 1,wherein the polymerase to be detected is a reverse transcriptase. 9.Method according to claim 8, wherein the reverse transcriptase is HumanImmunodeficiency Virus (HIV) reverse transcriptase.
 10. Method forscreening for the possibility of Human Immunodeficiency Virus (HIV) in asample comprising the steps of:a) preparing a lysate from a sample inwhich HIV is suspected to be present, b) adding to the lysate a templateribonucleic acid, and monodeoxyribonucleoside triphosphates to form amixture, wherein said monodeoxyribonucleoside triphosphates include atleast one detectable and at least one separate immobilizablemonodeoxyribonucleoside triphosphate, wherein said immobilizablemononucleoside triphosphate is not used as a detectable label, c)incubating the mixture to incorporate the detectablemonodeoxyribonucleoside triphosphate and the immobilizablemonodeoxyribonucleoside triphosphate into a newly formed nucleic acid,d) then transferring the mixture into a vessel which selectively bindsthe immobilizable monodeoxyribonucleoside triphosphate and the newlyformed nucleic acid in which the immobilizable monodeoxyribonucleosidetriphosphate is incorporated, e) separating any excess detectablemonodeoxyribonucleoside triphosphate from the vessel, and f) detectingany detectable mononucleotide bound to said vessel as an indication ofthe possibility of the presence of Human Immunodeficiency Virus (HIV).11. The method according to claim 10, wherein a primer is added to themixture before incubation.
 12. Method for determining the inhibitoryeffect of a substance on polymerase activity comprising the steps of:a)combining the substance with a polymerase in the presence of a templatenucleic acid and mononucleoside triphosphates to form an incubationmixture, wherein said mononucleoside triphosphates includes at least onedetectable and at least one separate immobilizable mononucleotidetriphosphate, wherein said immobilizable mononucleoside triphosphate isnot used as a detectable label, b) incubating said incubation mixture toincorporate the detectable mononucleoside triphosphate and theimmobilizable mononucleoside triphosphate into a newly formed nucleicacid, c) separating the newly formed nucleic acid from anynon-incorporated detectable mononucleoside triphosphates by contactingsaid newly formed nucleic acid with a solid phase which selectivelybinds the immobilizable mononucleoside triphosphate and the newly formednucleic acid in which the immobilizable mononucleotide is incorporated,and d) detecting the detectable mononucleoside triphosphate incorporatedinto the newly formed nucleic acid as an indication of the inhibitoryeffect of a substance on polymerase activity.
 13. Method for detectingan antibody to a polymerase in a sample comprising the steps of:a)combining the sample containing the antibody to be detected with atemplate nucleic acid, mononucleoside triphosphates and a polymerase toform an incubation mixture, wherein said mononucleoside triphosphatesinclude a detectable mononucleoside triphosphate and a separateimmobilizable mononucleoside triphosphate, wherein said immobilizalemononucleoside triphosphate is not used as a detectable label, b)incubating the incubation mixture to incorporate the detectablemononucleoside triphosphate and the immobilizable mononucleosidetriphosphate into a newly formed nucleic acid, c) contacting theincubation mixture with a solid phase which selectively binds theimmobilizable mononucleoside triphosphate including the immobilizablemononucleoside triphosphate which is incorporated in the newly formednucleic acid, d) separating the newly formed nucleic acid from anyremaining detectable mononucleoside triphosphate and e) detecting thedetectable mononucleoside triphosphate incorporated into the newlyformed nucleic acids as an indication of the presence of said antibody.14. Method for detecting a promotor sequence in a sample comprising thesteps of:a) combining a sample suspected of containing the promotorsequence to be detected, with a detectable mononucleoside triphosphate,a separate immobilizable mononucleoside triphosphate and a polymerase toform an incubation mixture, wherein said polymerase requires thepromotor sequence to initiate a polymerase reaction, and wherein saidimmobilizable mononucleoside triphosphate is not used as a detectablelabel, b) incubating said incubation mixture to incorporate thedetectable mononucleoside triphosphate and the immobilizablemononucleoside triphosphate into a newly formed nucleic acid, c)contacting the incubating mixture with a solid phase which selectively[and essentially completely]binds the immobilizable mononucleosidetriphosphate and the newly formed nucleic acid in which theimmobilizable mononucleoside triphosphate is incorporated, d) separatingthe newly formed nucleic acid from any remaining detectablemononucleoside triphosphate, and e) detecting the detectablemononucleoside triphosphates incorporated into the newly formed nucleicacid as an indication of the presence of a promotor sequence.